Synthetic nucleases such as meganucleases, zinc-finger nucleases (ZFNs), and TAL-effector nucleases (TALENs) are powerful and versatile tools for genome engineering to induce endogenous gene disruption, targeted gene addition, and chromosomal rearrangements in cells and organisms and thus are broadly useful in research, biotechnology, and medical fields.
The synthetic nucleases recognize a specific target nucleotide sequence and induce site-specific DNA double strand breaks (DSBs) in the genome, whose integrity is restored via endogenous DNA repair systems known as non-homologous end joining (NHEJ) and homologous recombination (HR), resulting in targeted mutagenesis and gene modification. In the absence of homologous donor DNA, DSBs are mainly repaired by NHEJ, a dominant repair system over HR in higher eukaryotic cells and organisms. Gene modification by HR is done by exact replication of the sequence of HR donor DNA, whereas NHEJ causes random gene modification. As NHEJ is intrinsically error-prone, small insertions and deletions (indels) may be generated at the DSB site, which then leads to genetic mutations by inducing frame-shift mutations.
Even though zinc-finger nuclease and TAL-effector nuclease are useful tools for designing a genetic modification in eukaryotic cells and organisms, a use thereof is highly limited. This is because in general, it is highly difficult to distinguish a mutant cell having a genetic mutation induced by a synthetic nuclease and a wild-type cell phenotypically, making it difficult to isolate mutant cells only.
In other words, one of the biggest roadblocks to apply synthetic nucleases in gene therapy and basic research is a lack of systems to enrich or select gene-modified cells. For example, the therapeutic efficacy of ZFNs that induce targeted disruption of the human chemokine receptor 5 (CCR5) gene which encodes a co-receptor of human immunodeficiency virus (HIV) largely depends on the number of CCR5-knockout cells induced by ZFNs. However, only a limited fraction of cells are mutated by the ZFNs and the remaining cells with at least one copy of the intact CCR5 gene will serve as hosts for HIV replication. Furthermore, laborious screening of numerous clones is often required to obtain gene-disrupted cells because only a minor fraction of cells are modified by nucleases. Also, even if CCR5-knockout cells can be selectively proliferated in vivo due to immunity against HIV infection, the enrichment of mutant cells prior to transplanting them will increase the potential therapeutic efficacy thereof.
Furthermore, a gene modifying function of synthetic nuclease allows the generation of gene-modified transformants, which can be applied to a large scale production of useful proteins and the treatment of incurable diseases. However, when producing transformed animals from large animals such as pigs and cows, a direct injection of ZFN mRNA into a fertilized egg at pronucleus stage has very low transformation efficacy due to mosaicism occurred during the generation of fertilized egg. Thus, as a reproduction method, nuclear transplantation, which injects the transformants generated by ZFN into an donor nucleus, is mostly used, and for this method, a large amount of transformants are required. For a large-scale production of transformants, an efficient method is required for selecting the transformants whose genes are modified by ZFN, through introducing a plasmid comprising ZFN DNA into the cell. Therefore, if a method for enriching or isolating the cells, whose target genes are modified by synthetic nuclease, in a high ratio can be established, it would be widely used in various areas where a synthetic nuclease can be applied.